This invention relates generally to biomarker screening and more particularly to identifying new targets for pharmacological inhibition.
This invention also relates generally to analytical (assays) methods for identifying compounds useful for promoting health in living mammalian systems. In particular this invention relates to assays and analytical tools for monitoring health in living mammals.
The function of complex living biological organisms relies on the meticulous control of cellular activity, including close regulation of cell growth, proliferation and function. The family of enzymes known as phospholipases A2 has been implicated in the control of cellular activity by catalyzing the esterolytic cleavage of fatty acids from phospholipids, thereby regulating the release of lipid second messengers, cellular growth factors, and the properties of the cellular membrane (Samuelsson et al., Annu. Rev. Biochem. 47:997-1029, 1978; Moolenaar, W. H., Curr. Opin. Cell. Biol. 7:203-10, 1995). In particular, by controlling the production of second messengers such as arachidonic acid and its biologically active eicosanoid metabolites, phospholipases A2 are involved in modulating such processes as cellular growth programs, inflammation, vascular tone and ion channel function. (Needleman et al., Annu. Rev. Biochem. 55:69-102, 1986).
Phospholipases A2 are a broad family of enzymes with varying kinetic and physical properties, and distinct functions. Early research focused on distinguishing broad classes of the enzymes within the larger family. Several classes were distinguished using in vitro activity assays, and are categorized based on the dependence of their enzymatic activity on the presence of calcium ion. (See e.g., Demel et al, Biochim. Biopliys. Acta 406:97-107, 1975). Thus, one class, the secretory phospholipases A2 are distinguished by an obligatory dependence on high (millimolar) concentrations of calcium ions, as well as low molecular weights (14-18 kDa) and relative heat stability. (Demel et al., supra; Tischfield, J. A., J. Biol. Chem. 272:17247-50, 1997). The activity of a second class, the cytosolic phospholipases A2 is facilitated by the presence of nanomolar concentrations of calcium ions, but the presence of the calcium ion is not obligatory. (Loeb et al., J. Biol. Chem. 261:10467-70, 1986; Kramer et al., Biochim. Biophys. Acta 878:394-403; Glover et al., J. Biol. Chem. 270:15359-67, 1986). A third class of enzymes is entirely calcium-independent in in vitro studies, and is also distinguished by a finely tuned inhibition by (E)-6-(bromomethylene)-3-(1-napthalenyl)-2H-tetrahydropyran-2-one (BEL). (Wolf et al., J. Biol. Chem. 260:7295-303; Hirashima et al., J. Neurochem. 59:708-14; Lehman et al., J. Biol. Chem. 268:20713-16).
Application of molecular biological techniques has provided some insights into the structure and function of founding members in each class of phospholipases A2 and has provided a further basis for distinguishing among the classes. (See, e.g. Demel et al., supra; Evenberg et al., J. Biol. Chem. 252: 1189-96, 1977; Tischfeld, J. A., J. Biol. Chem. 272: 17247-50, 1997). For example, members of the secretory phospholipases A2 use a calcium ion to polarize the carbonyl for attack by a histidine-activated H2O molecule, while the intracellular phospholipases use a nucleophilic serine. The calcium-facilitated phospholipases A2 have a GXSGS (SEQ ID NO: 2) consensus lipase motif, in contrast to the iPLA2 group which has a GXSTG (SEQ ID NO: 3) consensus motif. The calcium-independent phospholipases A2 are also distinguished by a consensus sequence for nucleotide binding. (Andrews et al., Biochem. J. 252:199-206, 1988; Tang et al., J. Biol. Chem. 272:8567-75, 1998). These findings have clearly boosted progress toward identifying the polypeptides responsible for catalyzing the synthesis of the eicosanoid metabolites and toward understanding the regulatory mechanisms of phospholipases A2 that are involved in normal and disease states.
The more recent developments of intense genome sequencing efforts have produced partial sequence data on the phospholipases and have led to related structural insights. For example, two new calcium-facilitated phospholipases have recently been described based on data from protein and nucleotide databases. (Underwood et al., J. Biol. Chem. 273: 21926-32, 1998; Pickard et al., J. Biol. Chem. 274: 8823-31, 1999). Further, during sequencing of the long arm of chromosome 7 in the Human Genome Sequencing Project, a predicted protein product of 40 kDa was identified. The polypeptide contained two 10 amino acid segments homologous to the lipase and nucleotide-binding consensus sequences described for the founding members of the iPLA2 family. (Tang et al., supra).
Earlier work has been done with respect to phospholipases and certain disease conditions in animals. For example, intensive study of reperfusion injury in myocardial tissue has led to the hypothesis that pathology is ultimately generated because of membrane phospholipid breakdown attributable to activation of myocardial phospholipase A2 activity. (See e.g. Van der Vusse et al., Hydrolysis of phospholipids and cellular integrity, In: H. M. Piper (ed.) Pathophysiology of Severe Ischemic Myocardial injury, Kluwer Academic Publishers, Dordrecht, The Netherlands, 1990, 167-93). Furthermore, calcium-dependent and calcium-independent phospholipase A2 activities have also been found to be present in the human cerebral cortex. Some reports have suggested a possible link between the activity of both calcium-dependent calcium independent phospholipases A2 and cortical degenerative diseases such as Alzheimer's disease. (For reviews, see e.g. Farooqui et al., Neurochem. Int. 30: 5 17-22, 1997; Farooqui et al., Brain Res. Bull. 49: 139-53, 1999).
Certain inhibitors of phospholipases A2 have been identified as possible therapeutic candidates for treating PLA2-mediated diseases. For example, fatty acid trifluoromethyl ketones, bromoenol lactone, methyl arachidonyl fluorophosphonate, benzenesulfonamides and other specific inhibitors of phospholipases A2 have been shown to decrease PLA2 activity and all have been considered for treating inflammatory diseases thought to be mediated by PLA2. (See e.g. Farooqui et al, 1999, supra). Nevertheless, as noted above, the phospholipases A2, as well as the iPLA2 subfamily itself, are a heterogeneous group of enzymes, with differing molecular weights, substrates, and responses to inhibitors. Because of this, the development of agents for treating diseases mediated by these compounds is ideally based upon determining and characterizing the structure and functional characteristics of the particular iPLA2 involved in the disease process. Thus, it is important to identify and characterize the phospholipases A2 family members.
During the last decade, excessive consumption of fat in high caloric Western diets in conjunction with a sedentary life style, has resulted in an epidemic of obesity in industrialized nations (1, 2). Obesity is associated with insulin resistance, hypertension, dyslipidemia, type 2 diabetes and atherosclerosis, which collectively constitute the metabolic syndrome (3, 4, 5). Despite the enormous proportions of this public health problem, the biochemical mechanisms underlying the metabolic syndrome and its end-organ sequelae are poorly understood.
With respect to diabetes, glucose utilization is necessary for the body to be able to use sugar which is stored in the blood as glucose. Insulin initiates the process of taking glucose from the blood and moving it into the cells. However, when glucose builds up in the blood instead of going into cells (e.g., insulin resistance), it can cause serious life threatening problems which results in type 2 diabetes. These include heart disease (cardiovascular disease), blindness (retinopathy), nerve damage (neuropathy), and kidney damage (nephropathy).
Type 2 diabetes is the most common form of diabetes. In this condition the body does not produce enough insulin to cause cells to transport glucose or the cells are not sensitive enough to the insulin present. The concentration of blood glucose becomes and remains high in the blood resulting in unnecessary and undesired damage to the body. Thus glucose is not utilized, proteins are covalently modified, inappropriate oxidation occurs and a change to fatty acid substrate occurs.